Brief Communication |
Characterization of Sox9 in European Atlantic Sturgeon (Acipenser sturio)
From the Department of Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany; Auf dem Schnee 54, 58313 Herdecke, Germany
Address correspondence to A. Ludwig at the address above, or e-mail: ludwig{at}izw-berlin.de.
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Abstract |
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The Sox9 gene of Acipenser sturio, one of the most primitive vertebrates, was analyzed. No sex-specific differences were observed. Sturgeon Sox9 consists of three exons and two introns with completely conserved exon-intron boundaries showing high levels of homology to other vertebrate Sox9 sequences, especially in the N-terminus region containing the HMG box. We found strong evidence for negative (purifying) selection. In contrast to previous studies of other fishes, we observed no evidence for gene duplication in sturgeon. Phylogenetic analyses of Sox9 evolution revealed a basal position for sturgeon Sox9.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Genes of the Sox gene family encode evolutionary conserved transcription factors containing a 79-amino-acid-long, DNA-binding domain called high mobility group (HMG) box (Sinclair et al. 1990) sharing, by convention, at least 50% identical residues with the HMG box of the mammalian testis determining factor SRY (Pevny and Lovell-Badge 1997). Genes of the Sox family have been identified in a number of vertebrates and invertebrates, and at least 20 members are currently recognized in mouse and human (Schepers et al. 2002).
In addition to SRY, Sox9 is another member of this family involved in sex determination. Sox9 was originally identified as the gene responsible for the human disease campomelic dysplasia, which causes skeletal malformation and is associated with XY male-to-female sex reversal (Foster et al. 1994; Wagner et al. 1994). Sox9 expression is highly upregulated in the developing male genital ridges of mice, birds, and turtles (Kent et al. 1996; Morais da Silva et al. 1996; Spotila et al. 1998). In mice, it controls the Müllerian inhibiting substance (Mis) regulation (Arango et al. 1999). The conservation of male-specific expression of Sox9 led to the suggestion that it is involved in normal sex determination in all vertebrates (Kent et al. 1996; Graves 1998).
We investigated Sox9 in A. sturio—a member of Acipenseridae, which is considered one of the most primitive groups of fishes, which first appeared approximately 200 MYA, and, together with three other major lineages (polypteryforms, lepisosteids, and Amia), has been accepted as occupying the most basal positions below the teleosts within the actinopterygian phylogeny (Bemis et al. 1997). A. sturio is considered highly endangered, with the last remaining natural population in the Gironde River, France. There were only three natural reproductions since 1980. Artificial reproductions and stocking efforts are necessary to conserve this population. Because of their long life cycle, their late maturity (reached between an age of 15 and 20 years), and the absence of morphological sex-specific features, we searched for genetic markers for sex differentiation. Such markers would allow sex-specific rearing and therefore increase the chance of successful artificial reproduction.
The mechanisms of sex determination are poorly characterized in fishes and vary considerably, ranging from environmental sex determination and different forms of hermaphroditism to classical sex chromosomal XX/XY or WZ/ZZ systems. The gene responsible for sex determination has recently been discovered in the medaka fish, Oryzias latipes (Matsuda et al. 2002), but it is absent in other, and even closely related, fish species (Kondo et al. 2003). The presence of heteromorphic sex chromosomes has never been reported in sturgeon. However, a female heterogametic sex-determination pathway was suggested based on experiments with gynogenetic females (Van Eenenaam et al. 1999). We characterized the Sox9 gene in male and female European Atlantic sturgeon as one possible candidate gene for sex determination and also sought to gain more information about the evolution of this gene in general.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Specimens of A. sturio were obtained from individuals caught in the Garonne River and the Gironde River estuary in 1995 within the scope of the conservation program from the CEMAGREF, France. Both individuals were mature and used for artificial reproduction as described by Williot et al. (2000).
Total genomic DNA was extracted from ethanol-preserved fin tissue using the QIAamp Tissue Kit (Qiagen) following the manufacturer's protocol. A fragment was generated with the following primers: Sox9-For1 (5'-ATGAATCTCCTVGACCCCTWCMTGA-3'); Sox-Rev1361 (5'-ATG GGG GTG TAC ATG GGW CKC TG-3'). The primers were based on Takifugu rubripes Sox9 mRNA (AF329945). PCR mixtures contained 0.8 U AmpliTaq DNA Polymerase (Perkin Elmer), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2 200 µM dNTPs, 10 pmol of each primer, and 100–500 ng of DNA in a final volume of 25 µl. PCR cycling conditions were 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s. PCR products were purified (PCR purification kit; Qiagen) and bidirectionally sequenced with various internal primers using cycle sequencing and then analysed using an ABI Prism 3100 sequencer (ABI).
The identity of the obtained sequence was confirmed using BLAST analysis. The coding nucleotide sequence was aligned to the following additional vertebrate Sox9 sequences: Monopterus albus Sox9a1 and Sox9a2 (AF378150–1), Gasterosteus aculeatus Sox9a and Sox9b (AY351914–5), Danio rerio Sox9a and Sox9b (AY09934, AF277097), T. rubripes Sox9a and Sox9b (AY277964, AY277965), Mus musculus (BC023808), Homo sapiens (NM000346), Gallus gallus (U12533), Xenopus laevis (AY035397), Oncorhynchus mykiss Sox9a and Sox9b (AB006448, AF209872), Alligator mississippiensis (AF106572). Basic sequence statistics and genetic distances were performed using MEGA version 2.1 (Kumar et al. 2001). We carried out two types of phylogenetic analysis to investigate evolutionary relationships using PAUP* 4.0b10 (Swofford 2002): (1) neighbor-joining (NJ) based on LogDet/paralinear distances, which were designed to deal with unequal base frequencies in each pairwise sequence comparison and thus allow base compositions to vary over the tree (Lockhart et al. 1994); and (2) maximum-likelihood (ML) analysis. Before the ML analyses, we used likelihood ratio tests and the computer application MODELTEST 3.06 (Posada and Crandall 1998) to determine the model best suited to describe sequence evolution. Heuristic ML searches were performed with 10 replicates of random sequence addition and TBR branch swapping. Nonparametric bootstrap analyses with 100 pseudo-replicates were performed to obtain estimates of support for each node of the ML tree; NJ bootstraps employed 1,000 iterations.
The relative rates of synonymous (ds) and nonsynonymous (dn) substitutions were determined using the modified Nei and Gojobori (1986) method and corrected for multiple hits (Jukes and Cantor 1969) using MEGA 2.1, which was also used to perform a codon-based Z-test of selection by comparing dn and ds as detailed in Nei and Kumar (2000).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Sox9 gene was sequenced in male and female A. sturio (Acc. AY788912). No sex-specific differences were observed. The structure of Sox9 is that of a typical transcription factor with discrete DNA binding and transcriptional activation domains. The deduced amino acid sequence of 432 amino acids was aligned to Sox9 of different vertebrate species. The alignment is available on the webpage of the Institute for Zoo and Wildlife Research, Berlin (www.izw-berlin.de). As in other vertebrates, sturgeon Sox9 consists of three exons interrupted by two introns with completely conserved exon-intron boundaries (Figure1). In comparison to human Sox9, in A. sturio, the 41 residues PQA motif is replaced by a stretch of five glutamine residues. Sturgeon's Sox9 showed high levels of similarity to other vertebrate Sox9 sequences, especially in the N-terminus region containing the HMG box (100% identity with the HMG box of other fishes), where only two amino acid positions (47 and 79 of HMG box) were variable in all compared species. This region is responsible for DNA binding and contains signals for nuclear import that can direct the proteins into the nucleus (Südbeck and Scherer 1997). As in other vertebrate Sox9 genes, the GC content in sturgeon Sox9 was elevated (57.4%).
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The pattern of nucleotide substitution was compatible with that expected under the influence of past or contemporary negative (purifying) selection, as the rate of nonsynonymous substitutions per nonsynonymous sites (dn) was significantly lower than that of synonymous substitutions per synonymous sites (ds) observed among alleles at the DNA binding and transcriptional activation domain of the Sox9 gene (Table 1). The calculations of synonymous versus nonsynonymous nucleotide substitutions also produced evidence for strictly negative selection of both Sox9 copies in other fish species (Table 2).
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Both phylogenetic analyses yielded similar topologies (only the ML tree is shown in Figure 2). In the gene tree A. sturio, Sox9 occupies an intermediate position between two main lineages leading to the teleost fishes and the tetrapods (amphibian, reptiles, birds, and mammals) consistent with the established taxonomic relationship among vertebrates. In some fishes like rice field eel, zebrafish, stickleback, pufferfish, and rainbow trout, two copies of Sox genes were found (Chiang et al. 2001; Cresco et al. 2003; Koopman et al. 2004; Takamatsu et al. 1997; Zhou et al. 2003). The karyotype of the Acipenseridae is characterized by a very high chromosome number subdivided into three groups, with
120, 240–260, and 500 chromosomes, respectively. A. sturio has a chromosome number of 2n = 121 ± 3 (Tagliavini et al. 1999). Some authors believe that all species with 120 chromosomes are tetraploid (e.g., Ohno et al. 1969; Dingerkus and Howell 1976; Birstein and Vasiliev 1987; Birstein et al. 1997); others call them functionally diploids (Fontana 1994; Fontana et al. 1998a,b; Jenneckens 1999; Tagliavini et al. 1999; Ludwig et al. 2001). Despite the high chromosome number in A. sturio, we found only one Sox9 copy using our specific primers. This is similar to the study of medaka, in which only a single Sox9 copy was found (Yokoi et al. 2002). Possible explanations are that the duplication event occurred more recently in fish evolution or that the second copy was lost.
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Following the classical hypothesis of gene duplication, retention of both gene copies has a very low probability and would happen only if one of the two duplicated forms acquired a new, positively selected function (Cooke et al. 1997; Sidow 1996; Walsh 1995). However, analysis of previously published sequences produced no evidence for positive selection of Sox9 copies. More recently, a new theory explaining the high empirical retention rate of duplicated gene copies was proposed. Instead of the acquisition of novel functions, the partitioning of ancestral subfunctions among descendant gene duplicates by the reciprocal neutral fixation of degenerative regulatory mutations can contribute to permanent preservation of both copies (Force et al 1999; Hughes 1994; Stolzfus 1999). Cresco et al. (2003) proposed this to be the case for Sox9 in teleost fish, which was probably duplicated during the ray-fin fish genome duplication more than 300 million years ago (Taylor et al. 2001). They studied the expression of Sox9a and Sox9b in stickleback and zebrafish and found that embryos of both species showed largely similar expression patterns for Sox9a and Sox9b and that these functions, taken together, represent the functions executed generally by a single Sox9 ortholog in tetrapods. Our data, which shows that even the duplicated Sox9 copies were negatively selected, supports the hypothesis of subfunction partitioning between the two Sox9 duplicates, which were strongly conserved throughout evolution in the genomes of various fish species.
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Acknowledgments |
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We thank Eric Rochard (CEMAGREF) for providing samples; Dietmar Lieckfeldt and Anke Schmidt for technical support; and Jörns Fickel, Rob DeSalle, and two anonymous reviewers for helpful comments on the manuscript. This study was supported by a grant from the DFG (LU 852/2).
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Footnotes |
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Corresponding Editor: Rob DeSalle
Received April 24, 2004
Accepted July 20, 2004
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References |
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